Research on the stipe cracking of wine-cap mushroom (Stropharia rugosoannulata) in different humidity conditions

Stropharia rugosoannulata is a well-renowned edible mushroom due to its nutritional and nutraceutical properties. This article focuses on the study of stipe cracking in S. rugosoannulata, a common issue in outdoor cultivation of this mushroom in South China. The findings reveal that the stipe cracks of S. rugosoannulata are primarily horizontal (transverse). Typically, cracks appear between the annulus and the middle part of the stipe prior to the opening of the pileus. Following the opening of the pileus, a fresh crack appears on the upper part of the stipe above the annulus. During the growth of S. rugosoannulata, two distinct elongation sections are observed in the stipe, separated by the annulus. The location of cracks coincides with these elongation sections, and the sequence of crack occurrences matches with the sequence of these elongation sections. The frequency of stipe cracking varies according to developmental stages and humidity conditions. The conclusion of this study is that S. rugosoannulata stipes crack during elongation and within elongation sections when humidity is low (≤ 60%), with the S3 developmental stage having the highest risk of cracking.


Research on the stipe cracking of wine-cap mushroom (Stropharia rugosoannulata) in different humidity conditions
Lei Huang 1,2 , Can Si 1 , Hongyu Shi 1,2 , Chunmei He 1* & Jun Duan 1* Stropharia rugosoannulata is a well-renowned edible mushroom due to its nutritional and nutraceutical properties.This article focuses on the study of stipe cracking in S. rugosoannulata, a common issue in outdoor cultivation of this mushroom in South China.The findings reveal that the stipe cracks of S. rugosoannulata are primarily horizontal (transverse).Typically, cracks appear between the annulus and the middle part of the stipe prior to the opening of the pileus.Following the opening of the pileus, a fresh crack appears on the upper part of the stipe above the annulus.During the growth of S. rugosoannulata, two distinct elongation sections are observed in the stipe, separated by the annulus.The location of cracks coincides with these elongation sections, and the sequence of crack occurrences matches with the sequence of these elongation sections.The frequency of stipe cracking varies according to developmental stages and humidity conditions.The conclusion of this study is that S. rugosoannulata stipes crack during elongation and within elongation sections when humidity is low (≤ 60%), with the S3 developmental stage having the highest risk of cracking.
Stropharia rugosoannulata, also known as Wine-cap mushroom or Roundhead mushroom, has a robust fruiting body.When mature, the stipe grows to a length of 9-15 cm and generates a deeply grooved annulus (ring) after the pileus opens 1 .
S. rugosoannulata is not only a food source high in protein, low in fat, and rich in minerals, vitamins, and dietary fiber 2 , but also a high-quality raw material for extracting bioactive compounds such as polysaccharides, sterols, and flavor peptides [3][4][5] .Additionally, it efficiently degrades and transforms various types of agricultural and forestry waste during its growth process 6 , helping to eliminate environmental pollutants and improve soil fertility [7][8][9] .S. rugosoannulata only needs remarkably simple cultivation techniques, yet it results in high yields 10 .Due to its significant application value in many fields, including dietary nutrition improvement, functional food production, environmental pollution control, ecological agriculture development, agricultural and forestry waste utilization, and rural poverty alleviation, S. rugosoannulata is recommended for cultivation by the Food and Agriculture Organization of the United Nations 11 .
S. rugosoannulata was first domesticated and cultivated in Germany in 1969.Subsequently, it was introduced and cultivated in other European countries such as Poland, Czechoslovakia, Hungary, and the Netherlands.By 1983, it was introduced to the United States 1,10 .China introduced a strain of S. rugosoannulata from overseas in 1992 and the cultivation of S. rugosoannulata began to spread ever since 12 .Since 2013, the cultivation of S. rugosoannulata has been vigorously promoted as a poverty-reducing project in various regions of China, leading to a rapid growth in cultivation area and production.In 2021, China's yield of fresh S. rugosoannulata mushroom exceeded 210,000 tons, marking a 43% increase from the yield recorded in 2019 13 , Simultaneously, the cultivation area expanded to over 4000 hectares 14 .
In South China, S. rugosoannulata is suitable for outdoor cultivation in winter.It can be cultivated from December to March of the following year 15 .In cultivation practices in Guangdong province, large-scale cracking of S. rugosoannulata stipes is often observed, which significantly compromises the integrity of mushroom appearance, thereby diminishing its quality and commercial value.
The cracking of mushroom pileus in response to low humidity is a common phenomenon 1 .Mushrooms with thick and long stipes, such as Boletus sp., Armillaria sp., Amanita sp., Pleurotus eryngii, and S. rugosoannulata, can also experience stipe cracking in the wild.However, there is limited research on the phenomenon of mushroom cracking, with only reported in Lentinula edodes (Shiitake mushroom) the induction of pileus cracking to form flower-like pileus 16 , while studies on stipe cracking have not been reported thus far.
This article aims to investigate the number and location of stipe cracks in S. rugosoannulata under different humidity conditions, and to correlate these observations with the elongation process of the stipe during different developmental stages.A diagrammatic model is developed to illustrate stipe cracking, and the mechanisms behind this phenomenon are explained.The article further provides specific humidity conditions that can be applied to the factory cultivation of S. rugosoannulata mushrooms.

S. rugosoannulata
S. rugosoannulata involved in this research are identified by Professor Jun Duan from the South China Botanical Garden of the Chinese Academy of Sciences as fruiting bodies of the strain "Zhongke No. 1 Red Songrong", which is a new variety of S. rugosoannulata bred through systematic selection by the South China Botanical Garden of the Chinese Academy of Sciences and preserved in the laboratory of this research institution.They were produced by the understory bed cultivation method and the cultivation substrate formulation consisted of 40% rice husk, 40% sawdust, and 20% shredded forest litter.Fallen leaves, with a thickness of approximately 5 to 10 cm, served as the casing layer.The spawn was sowed in early November 2022, and mushrooms were harvested from mid-December 2022 to late March 2023.All methods were carried out in accordance with relevant guidelines.
The growth and development process of S. rugosoannulata mushroom can be divided into 5 stages in chronological order 17 .In Stage 1 (S1, young stage), the mushroom has a short and stout stipe.In Stage 2 (S2, early elongating stage), the stipe begins to elongate.In Stage 3 (S3, mid elongating stage), the stipe significantly elongates, but the pileus remains unopen, and no ring structure is visible.In Stage 4 (S4, half-open stage), the pileus starts to open, with the lower edge detaching from the stipe, but it is not fully expanded, taking on a roundhead shape.A ring appears, and the upper part of stipe is exposed.In Stage 5 (S5, fully open stage), the stipe grows to its maximum length and finishes its elongation, and the pileus extends horizontally to the top, releasing spores (Fig. 1).

Measurement of stipe cracks in S. rugosoannulata
Samples of S. rugosoannulata mushrooms of all developmental stages with consistent morphology were harvested, with a minimum sample size of 30 individuals per stage.The length of stipes and the location of cracks (the distance from cracks to the base of stipes) were measured.The stipe cracking rate (= number of stipes with cracks / number of stipes × 100%) of each stage were calculated and the maximum number of cracks on each individual stipe were counted.Additionally, relative humidity (RH) and air temperature at the time of harvest were recorded.

Measurement of stipe elongation in S. rugosoannulata
After the young mushrooms emerged on the surface of the cultivation substrate, they were transferred, along with the base substrate, into pots.These pots were then placed indoors, providing a controlled environment

Effects of casing layer on the cracking of S. rugosoannulata stipes
A cultivation plot with dense mushroom growth was selected and divided into two equal halves.The casing layer of one half was removed, exposing the stipes of S. rugosoannulata mushrooms completely to the air.In the other half, the thickness of the casing layer was increased to 15 cm, burying the mushrooms completely within the casing layer.On both of the aforementioned plots, ten individuals in consistent growth status from various developmental stages were selected, and the cracking of stipes was recorded one day after.The experiment was conducted separately on three different dates at RH of 40%, 55%, and 70%.The cracking rate of S. rugosoannulata stipes in different developmental stages was calculated, and the RH inside the casing layer was recorded.

Statistical analysis
Statistical analyses were performed using Excel 2013.Data with repeats were presented as the mean ± standard deviation.The illustration diagram of stipe cracks and elongation process were created by Microsoft Office 2013.

Morphology of cracks on S. rugosoannulata stipes
During the development of S. rugosoannulata mushrooms, it is common for stipes to experience cracking (Fig. 2).The main type of cracks observed on the stipes of S. rugosoannulata is horizontal (transverse).Some horizontal cracks can encircle the stipe, forming circular cracks (Fig. 2c,d), while others may only occur on one side of the stipe (Fig. 2a,b).In certain cases, transverse cracks on the mushroom stipes may give rise to multiple longitudinal cracked pieces that outwardly curl.These segments are arranged in a radial pattern with two distinct levels (Fig. 2e-g).Some stipes may exhibit two parallel cracks at different heights (Fig. 2d).In other cases, new cracks may occur within an existing crack, resulting in overlapping positions of the two cracks (Fig. 2f).The outer layer of the cracked portion of the stipe appears harder and darker in color, while the interior of the cracks and the uncracked portions have a softer texture and a lighter color (Fig. 2f).The location where cracks occur on the stipe of S. rugosoannulata mushrooms is typically on the portion of the stipe that extends beyond the casing layer and exposes to the air (Fig. 2).At the unopen stages, the cracks typically occur in the upper-middle portion of the stipe below the annulus.The distance from the crack to the base of the stipe ranges from 3.0 to 9.0 cm.The maximum number of cracks is two, and they are parallel with a distance of approximately 1.0 to 2.0 cm (Fig. 3).At the open stages, there may also be one crack on the upper part of stipe, approximately 0.5 to 1.5 cm above the annulus (Fig. 4).The number of transverse cracks on an individual stipe range from one to two.

Cracks on S. rugosoannulata stipes in different developmental stages
During the development process of S. rugosoannulata, the stipes elongated by nearly 9 cm (Table 1).In S1, no cracks were observed on the stipes.However, in the stages from S2 to S5, cracks occurred on the stipes.In S2, the maximum number of cracks observed was one.In S3, the number of cracks could increase by one, with a maximum of two cracks.These cracks were located below the annulus.The occurrence of cracks on the stipes in S4 was similar to S3.In S5, the stipe may crack once above the annulus, but only on stipes that have experienced a maximum of 1 crack below the annulus.In S4 and S5, the maximum number of cracks observed on a stipe was two (Fig. 5, Table 1).
The cracking characteristics of S. rugosoannulata stipes are summarized as follows: Stipe cracking was observed to start from S2, but the cracking rate was lowest in this stage.As the stipes elongated, the cracking rate increased, reaching its maximum in S5.After S5, the cracking rate remained stable.There was a significant increase in the cracking rate during the transitions from S2 to S3 and from S4 to S5, and the increase during S2 to S3 transition was greater.Among all the stages, S3 had the highest likelihood of stipe cracking (Fig. 6).Meanwhile, S3 is also the optimal harvesting stage for S. rugosoannulata mushrooms as they approach commercial   www.nature.com/scientificreports/maturity in this stage 17 .This indicates that stipe cracking has a significant destruction to the commercial value of mushrooms.

Environmental conditions influencing the cracking of S. rugosoannulata stipe
When RH of the cultivation field was ≤ 60%, cracks occurred on S. rugosoannulata stipes.When the RH dropped to around 20%, the cracking rate of the stipes reached nearly 100%.Conversely, when the RH of the cultivation Location of annulus/cm 2.4 ± 0.2 4.0 ± 0.3 9.1 ± 0.9 9.2 ± 0.9 9.4 ± 0.9 Location of cracks/cm -3.1 ± 0.  www.nature.com/scientificreports/field was ≥ 62%, cracks was not observed on S. rugosoannulata stipes (Fig. 6).RH of the cultivation field showed a negative correlation with the cracking rate of S. rugosoannulata stipes (Fig. 7a).Stipe cracking occurred in a temperature range of 11-23 °C, and there was no significant correlation between the cracking rate and temperature of the cultivation field (Fig. 7b).

Elongation process of S. rugosoannulata stipes
When cultivated at 24 °C and 70% RH, S. rugosoannulata stipes increased from an average height of 4.2 cm to 13.1 cm within a period of four days.And after the fourth day, the stipe no longer elongated (Fig. 8a).Each segment marked from 0 to 4 exhibited elongation, but the elongation of S. rugosoannulata stipe primarily occurred in the upper-middle portion of the stipe, especially in the portion covered by the pileus (segment 0) (Table 2).When cultivated at 24 °C and 70% RH, the stipe inside the pileus experienced three instances of elongation over a period of three days, with daily increments of 2.3 cm, 1.3 cm, and 1.0 cm respectively.Three elongation sections were located at specific distances from the base of the stipe: 4.2-6.5 cm, 6.5-7.8 cm, and 10.0-11.0cm.Elongation process of the stipe was divided into two periods based on the occurrence of the annulus (the opening of the pileus).Before the annulus appeared, the stipe underwent elongation below it, resulting in the formation of the first and second elongation sections.These two sections were connected, forming a continuous elongation section.After the annulus appeared, the stipe below the annulus stopped elongating, while the upper part of stipe above the annulus continued to elongate, resulting in the occurrence of another elongation section at the top of the stipe.The elongation section of the stipe after the pileus opening is separated from the elongation section before the pileus opening by the annulus, creating two independent elongation sections of the stipe (Fig. 8b).
By comparing the locations where S. rugosoannulata stipes cracked and elongated, it can be discovered that these two locations largely coincide with each other, both in specific section and in chronological order (Figs. 5  and 8b).

Elongation and cracking of postharvest S. rugosoannulata stipes
Postharvest S. rugosoannulata mushrooms were still capable of completing the growth and development process from S2 to S5 (Fig. 9), with the stipe undergoing a noticeable elongation of 3.2 cm.However, during the entire elongating process, the stipes did not exhibit any cracking when the RH was low (40-60%) (Table 3).This suggests that maintaining a connection between the stipe and the substrate was a necessary condition for stipes to crack.

Effect of casing layer on stipe cracking in S. rugosoannulata
After the casing layer was removed, S. rugosoannulata stipes were exposed to the air.When the RH was 40% and 55%, mushrooms in the initial stages of S1, S2, S3 and S4 exhibited stipe cracking after one day of growth, while mushrooms in the initial stage of S5 did not show any stipe cracking.Conversely, when a thicker casing layer completely covered all the mushrooms, RH inside the casing layer increased to a range of 63% to 75%, significantly higher than the RH outside.In this situation, none of the S. rugosoannulata stipes experienced any cracking (Table 4).These results indicated that S. rugosoannulata stipe only cracks in the elongation stages, particularly when the RH is low.Once the elongation stopped, the stipe no longer cracks.Casing layer effectively increases the environmental humidity and prevents stipe cracking of S. rugosoannulata.
In summary, S. rugosoannulata stipe is probable to crack (mainly in the form of horizontal cracks) during the process of elongation when environmental RH is ≤ 60%.There are three locations where the stipe cracks: two are located in the upper-middle part of stipe below the annulus, and one is located in the upper part of stipe above the annulus.These cracking locations generally coincide with the elongation section of the stipe.Before the pileus opens, the stipe below the annulus elongates, and there may be one to two instances of cracking in the elongation section.After the pileus opens, the stipe below the annulus stops elongating, while the stipe above the annulus continues to elongate, and there may be one instance of cracking in the elongation section.Stipes that have experienced cracking twice below the annulus do not crack again above the annulus.Once the stipe has stopped elongating, no further cracking occurs, and postharvest S. rugosoannulata stipes do not exhibit any

Discussion
The mechanism of stipe cracking in S. rugosoannulata Turgor pressure is the pressure exerted by water-filled vacuoles against the cell wall, resulting in an outward force on the cell wall.Turgor pressure creates mechanical stress within the cell wall 18 .Mushroom maintains turgor  www.nature.com/scientificreports/pressure by absorbing water from the substrate through mycelium, which is one of the driving forces for stipe elongation.When mushroom cells absorb water, the turgor pressure increases 19 .In low RH (≤ 60%) environments, on one hand, the outer layer of the stipe undergoes dehydration and shrinks, becoming dry and rigid, probably forming micro-cracks 20 .On the other hand, as turgor pressure increases within the stipe, the stress exerted on the outer layer of the stipe also increases, causing the micro-cracks to widen and eventually develop into visibly noticeable cracks.The reason for the occurrence of two parallel cracks on the stipe may be related to the turgor pressure changing in the form of pulses during mycelial growth 21 .After being harvested, S. rugosoannulata no longer receives water supply from substrate, resulting in a decrease in turgor pressure within the stipe.As a result, cracking does not occur during the elongation process.Therefore, the external cause contributing to stipe cracking in S. rugosoannulata is mainly the low-humidity environment, while the internal cause is primarily the turgor pressure within the stipe.

Less cracks in reality than in theory
According to the model in Fig. 10, a fully developed stipe of S. rugosoannulata has three potential cracking locations.Theoretically, all three locations may crack, resulting in a maximum of three cracks.However, the results of this study indicate that on a single stipe, it is unlikely to observe horizontal cracks in all three potential cracking locations.Out of the three possible cracking locaitons, a maximum of two locations may exhibit cracking simultaneously, with a maximum of two instances of cracking occurring.Stipes that have experienced two instances of cracking below the annulus will not experience further cracking above the annulus.The most common occurrence is a single instance of cracking in one specific location of stipe below the annulus.The reason for the actual number of stipe cracking being lower than the theoretical number may be related to the release of turgor pressure within the stipe after the previous cracking occurs 22 .If the stipe has already experienced two instances of cracking, most of the turgor pressure within the stipe will be released, and the remaining pressure will not be sufficient to cause further cracking on the stipe.

Figure 1 .
Figure 1.Morphology of S. rugosoannulata mushrooms in different developmental stages (red arrows point to the annulus, yellow arrows point to the pileus, white arrows point to the stipe below the annulus, green arrows point to the upper part of stipe above the annulus).S1: young stage; S2: early elongating stage; S3: mid elongating stage; S4: half-open stage; S5: fully open stage.

Figure 3 .
Figure 3. Cracks on S. rugosoannulata stipes in unopen stages (red arrows point to the only one crack, yellow arrows point to the two parallel cracks).

Figure 5 .
Figure 5. Cracks on S. rugosoannulata stipes in different developmental stages (red arrows point to the cracks, black arrows point to the annulus, S1-S5: developmental stages in in chronological order).

Figure 6 .
Figure 6.Cracking rate of S. rugosoannulata stipes in different developmental stages and humidity conditions (RH: relative humidity).

Figure 7 .Figure 8 .
Figure 7. Environmental conditions influencing the cracking of S. rugosoannulata stipes (the blue dashed line represents the trend line, (a) RH, (b) temperature).Note: Data comes from S. rugosoannulata of S3.

Figure 10 .
Figure 10.Model of cracking on the elongating stipe of S. rugosoannulata (RH: relative humidity).

Table 1 .
Comparison of cracks on S. rugosoannulata stipes in different developmental stages.-means no existence; location means the distance to the base of stipe.

Table 2 .
Comparison of elongation of different segments of S. rugosoannulata stipe.

Table 3 .
Elongation and cracking rate of postharvest S. rugosoannulata stipes.The data of length was measured at 24 °C and 60% RH.

Table 4 .
Cracking rate of S. rugosoannulata stipes in different developmental stages with or without casing layer.RH means relative humidity.